Mass spectrometry (MS) is a powerful tool for the rapid and precise identification of proteins, either in an isolated, purified state or in situ in tissues and cells. Recent advances in protein molecular profile analysis and imaging mass spectrometry allow direct analysis and spatial localization of tumor specific biomarkers in thin tissue sections. Furthermore, MS techniques have been devised to study complex molecular interactions directly in cells, and thus elucidate patterns of signal transduction and other regulatory functions of proteins. Direct analysis of the whole proteome by mass spectrometry has the potential to rapidly address the global changes that control the heterogeneous biology of breast cancer. Therefore, we have chosen to address four important questions in this project: 1) the transition of in situ to invasive breast cancer, 2) the spatial correlation in tissues of anticancer drugs and surrogate markers predictive of response to therapy, 3) the identification of novel protein expression patterns that predict response to therapy, and 4) the identification of proteins in signaling complexes that are altered during breast cancer progression and which are targeted by molecular therapies. To address these questions we have formulated the following specific aims: Specific Aim 1. To determine the protein expression profiles of in situ and invasive breast cancers and to apply new methods of analysis for proteomics expression data that identify expression differences predictive of biologic behavior. Specific Aim 2. To determine if spatial profiling of anticancer agents in mammary tumors corresponds to target protein changes predictive of an antitumor response. Specific Aim 3. To discover by mass spectrometry novel proteins or protein profiles predictive of therapeutic response in mammary tumors. Specific Aim 4. To identify changes in signaling protein complexes induced by anti-signaling therapies using Direct Analysis of Large Protein Complexes (DALPC) and tandem MS.